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The occurrence of black gill syndrome (BGS) is a serious threat to the healthy culture of Eriocheir sinensis. Studying the innate immune ability of E. sinensis with BGS can help develop new strategies for disease prevention and treatment. Antimicrobial peptides (AMPs) have crucial roles in crustacean humoral immunity. In this study, we found that the expression levels of two antilipopolysaccharide factor (EsALF7 and EsALF-L), one Toll receptor 3 (EsToll3), and one Pelle kinase (EsPelle) were upregulated in E. sinensis with BGS. Moreover, ALFs expressions in E. sinensis with BGS were positively regulated by EsToll3 and EsPelle. The content of hydrogen sulfide (H 2 S) in the gills of E. sinensis with BGS was increased. Further studies showed that the expressions of cystathionine β-synthase (EsCBS) and cystathionine γ-lyase (EsCSE) in the gills of E. sinensis with BGS were upregulated, which positively regulate the production of H 2 S. Whether there was a correlation between the upregulation of ALFs expression and changes in H 2 S content? Further studies showed that 1) the expressions of EsToll3, EsPelle, EsALF7, and EsALF-L in the gills of E. sinensis were upregulated under H 2 S exposure and 2) the knockdown of EsCBS and EsCSE in E. sinensis reduced the transcriptions of EsToll3, EsPelle, EsALF7, and EsALF-L. To sum up, these findings suggest that upregulation of H 2 S content induced by CBS/CSE promotes the expression of ALFs through the Toll pathway in E. sinensis suffering from BGS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hepcidin belongs to a class of small cationic antimicrobial peptides rich in cysteine. It is synthesized by liver and is widely involved in host antimicrobial, antiviral and other immune responses. We identified and characterized three hepcidin genes (OpHep1, OpHep2 and OpHep3) in spotted knifejaw. All the OpHeps shared high identities with hepcidins in other teleost, containing alpha helix and β-sheets. Three OpHeps were all detected in healthy tissues, with the abundant expression in liver. They were significantly increased after Vibrio harveyi infection in the six immune-relevant tissues (liver, kidney, spleen, gill, skin and intestine). OpHeps knockdown in spotted knifejaw liver cells affected the mRNA levels of inflammation-related genes, including il1β, il6, il8, and nfκb. Further, the recombinant hepcidin proteins were effective in suppressing the growth of both Gram-negative and Gram-positive bacteria. To identify the function of OpHeps in vivo, we performed the overexpression of three OpHeps in zebrafish, and found OpHeps could significantly induce immune-related genes expression in transgenic zebrafish, including myd88, il10, il21, il16, tlr1, tlr3 and lysozyme. When infected with V. harveyi, OpHeps transgenic zebrafishes had a higher survival rate than wild-type zebrafishes. The expression of myd88, il10, il8, il1β, nfκb and lysozyme were all significantly up-regulated in transgenic fishes during bacterial infection. In summary, these results indicated that hepcidin could protect fish fight against pathogen through TLR/NFκB signaling cascade and Lysozyme. Three OpHeps would be potential targets for prevention of bacterial infections in aquaculture industry of spotted knifejaw, which provided a new idea for the molecular breeding of fish disease resistance., Competing Interests: Declaration of competing interest The authors read and approved this version of the final manuscript, confirmed the integrity of this work and declared no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ichthyophthirius multifiliis is a parasite that poses a considerable threat to aquaculture and the ornamental fish industry, but with limited effective treatment options available. This study employed RT-qPCR to detect and analyze the expression changes of partial toll-like receptor (TLR) genes (TLR1 and TLR21), adapter protein and signal transduction molecule genes (MyD88, TRIF, NF-κB, IRAK4, and IRF3), and cytokines (IL-6, IL-8, IL-13, CXC-α and CXCR1), as well as complement C3, in the skin, gill, fin, liver, head kidney and spleen of Rhinogobio ventralis under different infection conditions. Additionally, tissue sections and scanning electron microscopy were utilized to observe the pathological changes in the gills and fins of R. ventralis after infection with I. multifiliis. The expression patterns of TLR-related DEGs (differentially expressed genes) in diseased wild fish were analyzed, revealing upregulation of TLR1, TLR21, MyD88, NF-κB, IRAK4, TRIF, IRF3, IL-6, IL-8, IL-13, CXC-α, CXCR1, and C3 genes in various tissues, indicating that these genes may be involved in the immune response of R. ventralis to I. multifiliis infection. To further analyze the gene expression of sampled from the field, an artificial infection model of R. ventralis was established under laboratory conditions, with additional sampling from the skin and fins. These genes continued to show varying degrees of upregulation, but the results were not entirely consistent with those from Wudongde samples, which may be due to the more complex environment in the wild or differences in the degree of I. multifiliis infection in wild fish. The infection of I. multifiliis caused severe damage to the gills and fins of R. ventralis, characterized by extensive secretions on the gill and fin surfaces, with the presence of attached I. multifiliis trophonts, including damage and loss of gill filaments, swollen gill lamellae, and deformed gill plates, as well as cell proliferation and necrosis of gill epithelial cells. This study sheds light on the role of the TLR signaling pathway in resisting I. multifiliis infection and its associated histopathological changes in R. ventralis, providing valuable insights for the prevention and treatment of I. multifiliis infection in R. ventralis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Takifugu rubripes is a highly valued cultured fish in Asia, while pathogen infections can result in severe diseases and lead to substantial economic losses. Toll-like receptors (TLRs), as pattern recognition receptors, play a crucial role on recognition pathogens and initiation innate immune response. However, the immunological properties of teleost-specific TLR23 remain largely unknown. In this study, we investigated the biological functions of TLR23 (TrTLR23) from T. rubripes, found that TrTLR23 existed in various organs. Following bacterial pathogen challenge, the expression levels of TrTLR23 were significantly increased in immune related organs. TrTLR23 located on the cellular membrane and specifically recognized pathogenic microorganism. Co-immunoprecipitation and antibody blocking analysis revealed that TrTLR23 recruited myeloid differentiation primary response protein (MyD88), thereby mediating the activation of the ERK signaling pathway. Furthermore, in vivo showed that, when TrTLR23 is overexpressed in T. rubripes, bacterial replication in fish tissues is significantly inhibited. Consistently, when TrTLR23 expression in T. rubripes is knocked down, bacterial replication is significantly enhanced. In conclusion, these findings suggested that TrTLR23 played a critical role on mediation TLR23-MyD88-ERK axis against bacterial infection. This study revealed that TLR23 involved in the innate immune mechanism, and provided the foundation for development disease control strategies in teleost., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Myeloid differentiation factor-88 (MyD88) is a key adaptor of the toll-like receptor (TLR) signaling pathway and plays a crucial role in innate immune signal transduction in animals. However, the MyD88-mediated signal transduction mechanism in shellfish has not been well studied. In this study, a new MyD88 gene, CfMyD88-2, was identified in the Zhikong scallop, Chlamys farreri. The 1779 bp long open reading frame encodes 592 amino acids. The N-terminus of CfMyD88-2 contains a conserved death domain (DD), followed by a TIR (TLR/Interleukin-1 Receptor) domain. The results of the multi-sequence comparison showed that the TIR domain sequences were highly conserved. Phylogenetic analysis revealed that CfMyD88-2 was first associated with Mizuhopecten yessoensis MyD88-4 and Argopecten irradians MyD88-4. CfMyD88-2 mRNA was expressed in all scallop tissues, as detected by qRT-PCR, and the expression level was the highest in the mantle and hepatopancreas. In addition, CfMyD88-2 mRNA expression significantly increased after pathogen-associated molecular patterns (PAMPs, such as lipopolysaccharide, peptidoglycan, or polyinosinic-polycytidylic acid) stimulation. The results of the co-immunoprecipitation experiments in HEK293T cells showed that both CfMyD88-1 and CfMyD88-2 interacted with the TLR protein of scallops, suggesting the existence of more than one functional TLR-MyD88 signaling axis in scallops. Dual luciferase reporter gene assays indicated that the overexpressed CfMyD88-2 in HEK293T cells activated interferon (IFN) α, IFN-β, IFN-γ, and NF-κB reporter genes, indicating that the protein has multiple functions. The results of the subcellular localization experiment uncovered that CfMyD88-2 was mainly localized in the cytoplasm of human cells. In summary, the novel identified CfMyD88-2 can respond to the challenge of PAMPs, participate in TLR immune signaling, and may activate downstream effector genes such as NF-κB gene. These research results will be useful in advancing the theory of innate immunity in invertebrates and provide a reference for the selection of disease-resistant scallops in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-β (IFN-β) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-β (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

As a crucial member of pattern-recognition receptors (PRRs), the Tolls/Toll-like receptors (TLRs) gene family has been proven to be involved in innate immunity in crustaceans. In this study, nine members of TLR gene family were identified from the mud crab (Scylla paramamosain) transcriptome, and the structure and phylogeny of different SpTLRs were analyzed. It was found that different SpTLRs possessed three conserved structures in the TIR domain. Meanwhile, the expression patterns of different Sptlr genes in examined tissues detected by qRT-PCR had wide differences. Compared with other Sptlr genes, Sptlr-6 gene was significantly highly expressed in the hepatopancreas and less expressed in other tissues. Therefore, the function of Sptlr-6 was further investigated. The expression of the Sptlr-6 gene was up-regulated by Poly I: C, PGN stimulation and Vibrio parahaemolyticus infection. In addition, the silencing of Sptlr-6 in hepatopancreas mediated by RNAi technology resulted in the significant decrease of several conserved genes involved in innate immunity in mud crab after V. parahaemolyticus infection, including relish, myd88, dorsal, anti-lipopolysaccharide factor (ALF), anti-lipopolysaccharide factor 2 (ALF-2) and glycine-rich antimicrobial peptide (glyamp). This study provided new knowledge for the role of the Sptlr-6 gene in defense against V. parahaemolyticus infection in S. paramamosain., Competing Interests: Declaration of competing interest We declare that we have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) are one of the extensively studied pattern recognition receptors (PRRs) and play crucial roles in the immune responses of vertebrates and invertebrates. In this study, 14 TLR genes were identified from the genome-wide data of Octopus sinensis. Protein structural domain analysis showed that most TLR proteins had three main structural domains: extracellular leucine-rich repeats (LRR), transmembrane structural domains, and intracellular Toll/IL-1 receptor domain (TIR). The results of subcellular localization prediction showed that the TLRs of O. sinensis were mainly located on the plasma membrane. The results of quantitative real-time PCR (qPCR) showed that the detected TLR genes were differentially expressed in the hemolymph, white bodies, hepatopancreas, gills, gill heart, intestine, kidney, and salivary gland of O. sinensis. Furthermore, the present study investigated the expression changes of O. sinensis TLR genes in hemolymph, white bodies, gills, and hepatopancreas in different phases (6 h, 12 h, 24 h, 48 h) after stimulation with PGN, poly(I: C) and Vibrio parahaemolyticus. The expression of most of the TLR genes was upregulated at different time points after infection with pathogens or stimulation with PAMPs, a few genes were unchanged or even down-regulated, and many of the TLR genes were much higher after V. parahaemolyticus infection than after PGN and poly(I:C) stimulation. The results of this study contribute to a better understanding of the molecular immune mechanisms of O. sinensis TLRs genes in resistance to pathogen stimulation., Competing Interests: Declaration of competing interest We declare that we have no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that play a critical role in innate immune responses against pathogens. In the present study, a fish-specific TLR14 was identified and characterized from Monopterus albus (named MaTLR14), which consisted of a 2658 bp open reading frame encoding a protein of 885 amino acids. Phylogenetic analysis revealed that MaTLR14 belong to the TLR1 subfamily and shared the highest similarity to Paralichthys olivaceus TLR14. Immunohistochemistry assay showed that MaTLR14 mainly located in intestinal epithelial cells of hindgut. Immunofluorescence revealed that MaTLR14 largely localized to the intracellular region and partially co-localized with cell membrane of HeLa cells. The expression levels of MaTLR14 were upregulated in the liver, spleen, foregut and hindgut post infection with Aeromonas hydrophila. When stimulated with LPS and Flagellin, the MaTLR14 expression was elevated in isolated peripheral blood leukocytes. Further studies showed that recombinant MaTLR14-LRR could bind to both the gram-negative and gram-positive bacteria and cause agglutination. Subsequently, the signaling pathway of MaTLR14 was investigated. Confocal microscopy and co-immunoprecipitation assay demonstrated that MaTLR14 recruited MyD88 as adaptor. When overexpressed, MaTLR14 augmented the expression of TRAF6 and phosphorylation of ERK and p65, activated NF-κB and AP-1 and elicited the expression of il-6 and tnf-α. Collectively, MaTLR14 plays an important role in the microorganism recognition and signaling transduction., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The Manila clam (Ruditapes philippinarum), one of the major marine aquaculture species in China, is susceptible to infection with the pathogen Vibrio, which results in massive mortality and economic losses. Toll-like receptors (TLRs) are significant pattern recognition receptors (PRRs) of innate immunity that are involved in immune regulation against pathogenic invasion. Molecular characterization of Manila clam TLRs and investigations of their immune functions are essential to prevent and control Vibrio infection. In the present research, eight cDNA sequences of R. philippinarum TLRs (RpTLRs) were identified from previous transcriptome libraries and then classified into four groups, namely, P-TLR (one sequence), V-TLR (one sequence), Ls-TLR (two sequences) and sP-TLR (four sequences), based on the corresponding LRR domain arrangement of their protein structures within the typical TLR motifs. A selective pressure test firstly suggested that the molluscan P-TLR, V-TLR, Ls-TLR and sP-TLR families underwent positive selection, and different numbers of positive selection sites (PSSs) were identified in different domains of the four types of RpTLRs, as determined by PAML and analysis of website data. These findings indicated that the evolution of RpTLRs may be associated with their immune recognition and function. Furthermore, tissue-specific expression analysis showed that all RpTLRs were ubiquitously expressed in all test tissues and were dominant in hemocytes. Quantitative real-time PCR revealed that the cDNA expression of all eight RpTLRs was upregulated after injection with Vibrio anguillarum (P < 0.01) in R. philippinarum hemocytes, revealing that these RpTLRs play important roles in responding to pathogenic stimulation. In summary, these findings provide a foundation for future investigations of the molecular classification and evolutionary patterns of Toll-like receptors in invertebrates, and the innate immune responses of TLR signaling pathways in Mollusca., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Outbreaks of infectious disease in shrimp pose a serious threat to shrimp agriculture worldwide. Shrimp lack adaptive immunity and depend only on innate immunity as a defense system against infectious disease. Toll-like receptors (TLR) are reported to play a critical role in the innate immune system. In this study, we identified a Toll-like receptor gene of a species of freshwater shrimp, Macrobrachium nipponense, designated MnToll, for the first time. The sequence of MnToll encoded 935 residues arranged as 10 leucine-rich repeat (LRR) domains, a leucine-rich repeat C-terminal (LRR CT) domain and a Toll/interleukin-1 receptor (TIR) domain and displayed 90% amino acid similarity to previously identified TLRs (Toll 1 and 2) of Macrobrachium rosenbergii. We additionally evaluated mRNA expression of MnToll in various tissues, including heart, gills, stomach, digestive gland, ventral nerve cord, antennal gland and muscle. Following infection with a viral pathogen, white spot syndrome virus (WSSV), MnToll expression was significantly upregulated between 12 and 72 h. Our data collectively suggest that the newly identified MnToll gene belongs to the TLR family in shrimp and is potentially involved in innate host defense, especially against WSSV., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Toll-like receptors, which are a class of cell-surface proteins, have been regarded as the most important pattern recognition receptors in the innate immunity and play a vital role in multiple innate immune responses against pathogen invasion. The full-length cDNA of a novel Toll-like receptor (MrToll3) was identified from Macrobrachium rosenbergii in the current research. The nucleotide sequence of MrToll3 is 4481 bp long and contains a 3726-bp open reading frame encoding a putative protein of 1241 amino acids. MrToll3 was constitutively expressed in all the examined tissues, and high expression of MrToll3 was detected in gill, heart, and ganglion. The result of RNA interference assay revealed that silencing of MrToll1 remarkably suppressed the prophenoloxidase (proPO) expression and phenoloxidase (PO) activities while enhancing MrToll2 expression in the prawns. Furthermore, the expression of myeloid differentiation factor 88 (MyD88), anti-lipopolysaccharide factor (ALF) and crustin was remarkably down-regulated in the MrToll1-silenced prawns after white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge. MrToll2-silenced prawns exhibited the significant decline of ALF and crustin expression post the pathogen challenges, and silencing of MrToll3 obviously improved the immune deficiency (IMD) expression during the whole RNA interference assay. Additionally, higher mortality was observed in MrToll1-or MrToll2-silenced prawns after V. alginolyticus challenge, and the MrToll1-silenced prawns also showed the obviously enhanced susceptibility to WSSV. These results suggested that MrToll1, 2, and 3 were involved in the innate immune responses against WSSV and V. alginolyticus in M. rosenbergii., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) are important pattern recognition receptors (PRRs) of innate immune system, playing crucial roles in immune defense against pathogens. TLR18, a member of TLR1 family, is fish-specific TLR and involves in the immune response against bacterial infection. Currently, the structural biology of fish TLR18 is poorly elaborated. In this study, the structure and ligand binding of TLR18 (smTLR18) of soiny mullet (Liza haematocheila), an economically valuable aquaculture mugilid species, were analyzed. The extracellular domain (ECD) of smTLR18 formed an open-loop horseshoe-shaped structure with the concave surfaces made up of 19 parallel β-strands (LRR1-LRR19), lacking Z-loop that seen in human TLR9. The intracellular Toll/interleukin (IL)-1 (TIR) domain contained a central 4-parallel β-sheet (βA-βD) surrounded by 5 α-helices (αA-αE). Molecular docking analysis revealed that both ECD domain and TIR domain of smTLR18 could form homodimers. For the ECD homodimer, the main residues involved in dimer formation were located from LRR10 to LRR14. For the TIR homodimer, the residues involved in dimer formation were located in BB loop, αB helix, αC helix and DD loop. Ligand binding analyses revealed that peptidoglycans (PGNs) and lipopolysaccharides (LPS), two main bacterial pathogen-associated molecular patterns (PAMPs), were the potential ligands of smTLR18. The van der Waals and Coulombic interactions contributed to the interactions between smTLR18 and PGNs, while only van der Waals dominated the interactions between smTLR18 and LPS. The residues involved in ligands binding were located from LRR9 to LRR13. Our results provided the structural bases for elucidate the ligand binding of fish TLR18., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) represent a well-known family of conserved pattern recognition receptors the importance of which, in non-self recognition, was demonstrated in both vertebrates and invertebrates. Tunicates represent the vertebrate sister group and, as invertebrates, they rely only on innate immunity for their defence. As regards TLRs, two transcripts have been described and characterised in the solitary species Ciona intestinalis, referred to as CiTLR1 and CiTLR2. Using the Ciona TLR nucleotide sequences, we mined our available transcriptome of the colonial ascidian Botryllus schlosseri looking for similar sequences. We were able to identify a sequence, with similarity to CiTLR2 and, through in silico transduction and subsequent sequence analysis, we studied the domain content of the putative protein. The sequence, called BsTLR1, has a TIR and a transmembrane domain, four LLR and two LRR-CT domains. It is actively transcribed by both phagocytes and morula cells, the two circulating immunocyte types. In addition, we analysed bstlr1 transcription in vivo and in vitro, in different phases of the Botryllus blastogenetic cycle and under various experimental conditions. Our data show that there is a change in gene expression and mRNA location, according to the blastogenetic phase. Furthermore, we used a commercial antibody raised against the ectodomain of hTLR5 to study the possible functional role of Botryllus TLR(s). We observed that anti-hTLR5 significantly decreased in vitro phagocytosis and morula cell degranulation, two typical responses to the recognition of nonself. Collectively, our data add new information on the mechanisms of nonself recognition in a colonial ascidian., (Copyright © 2020. Published by Elsevier Ltd.)

The transcriptome of the caenogastropod mollusk Littorina littorea was scanned for the presence of sequences encoding Toll-like receptors (TLRs) and corresponding proteins involved in downstream TLR signaling pathway. In the transcriptomic snapshots of hemocytes and kidney tissues, 45 complete TLRs encoded by 35 genes were identified. Out of the 59 non-TLR molecules involved in a canonical TLR signaling pathway, 35 genes were classified as homologous and could be placed within the TLR-mediated MyD88-and MAPK-dependent circuitries. No reference vertebrate adapters TIRAP, TRIF and TRAM were identified in the transcriptome. The results of RNA-seq experiments with an immune challenge (rediae of the digenean Himasthla elongata) indicate that four TLRs (LlTLR1, 3, 5 and 8) and a set of upregulated genes involved in signal transduction (LlMyd88, LlTNFα, LlCASP8, LlFADD, LlNFKBIA (IkBα), LlIRAK1, LlSTAT1, LlMAPK14 (P38), LlMAP2K1 (MEK1/2), LlIRF3 and LlIRF5) may participate in the anti-digenean immune response of L. littorea., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Fatty liver injury (or disease) is a common disease in farmed fish, but its pathogenic mechanism is not fully understood. Therefore the present study aims to investigate high-fat diet (HFD)-induced liver injury and explore the underlying mechanism in fish. The tilapia were fed on control diet and HFD for 90 days, and then the blood and liver tissues were collected to determine biochemical parameter, gene expression and protein level. The results showed that HFD feeding signally increased the levels of plasma aminotransferases and pro-inflammatory factors after 60 days. In liver and plasma, HFD feeding significantly suppressed antioxidant ability, but enhanced lipid peroxidation formation, protein oxidation and DNA damage after 60 or 90 days. Further, the Nrf2 pathway and antioxidative function-related genes were adversely changed in liver of HFD-fed tilapia after 60 and/or 90 days. Meanwhile, HFD treatment induced apoptosis via initiating mitochondrial pathway in liver after 90 days. Furthermore, after 90 days of feeding, the expression of genes or proteins related to JNK pathway and TLRs-Myd88-NF-κB pathway was clearly upregulated in HFD treatment. Similarly, the mRNA levels of inflammatory factors including tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8 and IL-10 were also upregulated in liver of HFD-fed tilapia after 60 and/or 90 days. In conclusion, the current study suggested that HFD feeding impaired antioxidant defense system, induced apoptosis, enhanced inflammation and led to liver injury. The adverse influences of HFD in the liver might be due to the variation of Nrf2, JNK and TLRs-Myd88-NF-κB signaling pathways., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The Nile tilapia (Oreochromis niloticus) is one of the major food fish species produced in tropical and subtropical regions. However, this industry has been facing significant challenges from microbial infections. Understanding how hosts initiate immune responses against invading microbes is the first requirement for addressing disease outbreak prevention and disease resistance. Toll-like receptors (TLRs) are a family of evolutionarily conserved proteins that can recognize pathogen-associated molecular patterns (PAMPs). They thus play an essential role in innate immunity. TLR25 is a newly identified fish-specific member of the TLR1 subfamily. In this study, we investigate the molecular and functional characteristics of O. niloticus TLR25 (OnTLR25) via tissue expression patterns, gene expression modulation after challenge with bacteria and TLR ligands, subcellular localization in human and fish cells, and the signaling pathways TLR25 may induce. Transcriptional levels of OnTLR25 are high in immune-related organs such as the spleen and head kidney, and are increased following bacterial challenges. In addition, we show that OnTLR25 preferentially localizes to the intracellular compartment in transfected tilapia head kidney (THK) cell line. Furthermore, overexpression of the truncated form of OnTLR25 in THK cell line induced the expression of proinflammatory cytokines, such as tumor necrosis factor α, interleukin (IL)-1β, IL-8, IL-12a, and interferon-d2.13. Combined, our results suggest that TLR25 is likely to play an important role in the antimicrobial responses of the innate immune system of Nile tilapia., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Crustacean species are considered as a major sector in the aquaculture industry that plays a fundamental role in the world's economy. However, with a wide range of various epidemic diseases in the industry, studies of immune-related genes such as toll-like receptor genes are of great importance. Recently, the TLR in crustacean species has been described to perform a vital role in defense of crustacean against the pathogens. Meanwhile, many TLR genes from crustacean were characterized, and their contribution discovered in innate immunity against several pathogens. This review was aimed to present an overview of the crustacean TLRs including structural features that contained three major domains: a leucine-rich repeat (LRR) domains, a transmembrane area (TM), and a conserved region called Toll/interleukin-1 receptor (TIR) domain. The tissue distribution patterns of TLR genes, which act as a guide for future research on which TLR gene or genes that can be expressed, at which tissue or tissues. We also described recent works on the expression of the TLR gene that evaluated the immune function after pathogen stimulation in shrimp, crab, and crayfish. Furthermore, we recommended a prospective for future investigation plan that might contribute to the development and management systems in the global crustacean aquaculture industry. Lastly, we assumed that a clear understanding of the expression pattern and biological function of crustacean TLR genes could serve as a baseline for future immunological studies., (Copyright © 2020. Published by Elsevier Ltd.)

The double-Ig-IL-1R related molecule (DIGIRR) is a member of the TIR (Toll -Interleukin-1 receptor) superfamily and plays an important role in the immune system, it is also as a negative regulator of the IL-1 signaling pathway. In this study, we identified and characterized the miiuy croaker DIGIRR (mmi-DIGIRR) gene. The results of gene structure analysis indicated that there were differences between the mmi-DIGIRR and mammalian SIGIRR, which there were two immunoglobulin (Ig) domains contained in extracellular region of mmi-DIGIRR. Sequence alignment analysis showed that fish DIGIRR shared some conserved sequences with other vertebrates and the evolution was relatively conservative. In order to further validate the function of mmi-DIGIRR and its expression levels in various tissues of fish, qRT-PCR has been conducted. The results showed DIGIRR has significant expression levels in liver, skin and muscle while expression levels in heart are low. The LPS-induced NF-κB activation was inhibited by overexpression of DIGIRR significantly. In conclusion, the evolution and function of mmi-DIGIRR were comprehensively analyzed in this study, which would provide a theoretical basis for the future research of fish DIGIRR., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Tributyltin chloride (TBT-Cl) residual in water body had become a noticeable ecological problem for aquatic ecosystems. Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors that play key roles in detecting nonself antigens and immune system activation. In this study, we explored the effect of TBT-Cl exposure on four TLRs expression in river pufferfish, Takifugu obscurus. The four T. obscurus Toll-like receptors (To-TLRs) contained different types of domains such as leucine-rich repeats (LRRs), leucine-rich repeats, typical subfamily (LRR&#95;TYP) and other special domains. The To-TLRs mRNA transcripts expressed in all tissues, also To-TLR2 was investigated with higher level in kidney, as well as To-TLR3 in kidney, while To-TLR18 in liver and To-TLR22 in intestine. After the acute and chronic exposure of TBT-Cl, To-TLR2 and To-TLR3 mRNA transcripts were significantly down-regulated in gill. However, To-TLR18 and To-TLR22 were significantly up-regulated in gill and liver. Moreover, the histology and immunohistochemistry (IHC) results showed the different injury degrees of TBT-Cl in liver and gill and implied the cytoplasm reorganization after TBT-Cl stress and the function of immunoregulation for To-TLRs to TBT-Cl exposure. All the results indicated that To-TLRs might involve in sensing and mediating innate immune responses caused by TBT-Cl for keeping detoxification homeostasis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The functional spectrum of the teleostean head kidney covers haematopoietic, immune and endocrine signalling pathways with physiological effects that are likely to conflict if activated at the same time. An in vivo experiment on the salmonid fish maraena whitefish (Coregonus maraena) revealed that the head kidney shows a remarkably strong response after injection of Aeromonas salmonicida within 48 h. In order to investigate the potential influence of endocrine signalling on the initiation of immune responses, we established a primary culture of head-kidney cells of maraena whitefish. For the characterisation of this model system, we used flow cytometry complemented with an extensive panel of immunological/haematological and stress-physiological/neuroendocrinological qPCR assays. More than one third of the cells expressed the characteristic signature of myeloid cells, while more than half of the cells expressed those genes typical for lymphocytes and monocytes. In parallel, we quantified the expression of genes encoding endocrine receptors and identified ADRA2D as by far the most highly expressed adrenergic-receptor gene in head-kidney cells. The stimulation of the head-kidney cells with toll-like receptor ligands induced the expression of typical immune genes (IL1B, CXCL8, TNF, SAA) after only 1 h. The incubation with the stress hormones cortisol, adrenaline and noradrenaline also had an immune-activating effect, though less pronounced. However, cortisol had the strongest suppressive effect on the stimulation-induced immune response, while adrenaline exerted a comparably weaker effect and noradrenaline was almost ineffective. Moreover, we found that cortisol reduced the expression of genes coding for adrenergic and some glucocorticoid receptors, while noradrenaline increased it. In conclusion, the primary head-kidney cells of maraena whitefish reflect the immunological and neuroendocrinological diversity of the entire organ. This in vitro system allowed thus identifying the correlative changes between the activities of hormones and immune factors in salmonid fish in order to contribute to a better understanding of the regulation circuit between stress and immune defence., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Interleukin-1 receptor associated kinase (IRAK1) is one of the crucial signal transduction mediators in TLR/IL-1R signaling pathways in host immune system. To investigate about it in rohu (Labeo rohita), one of the economically important freshwater fish species in the Indian subcontinent, we cloned, characterized and analyzed its expression following bacterial infection and pathogens associated molecular patterns (PAMPs) stimulation. The full-length cDNA of rohu IRAK1 (LrIRAK1) consisted of 2765 nucleotide (nt) having an ORF of 2115 nt encoding a polypeptide of 704 amino acids (aa) with a molecular mass of 70.4 kDa. Structurally, LrIRAK1 consisted of twenty-nine helix, twelve strands and forty one coils making one N-terminal death domain (19-94 aa) and a central serine threonine kinase catalytic domain (or kinase domain) (188-489aa). In addition to these two prominent domains, LrIRAK1 also contained highly conserved amino acids viz., lysine 215 and aspartic acid 314 and threonine 185, 361 which were reported to be important for kinase and phosphorylation activity respectively in other animals. Similar to higher vertebrates, LrIRAK1 also consisted of CDK1 (cyclin-dependent kinase1) at 338-352 aa; NEK2 (NIMA-related kinase 2) at 47-61 aa; NEK6 (NIMA-related kinase 6) at 581-595 aa and AMPK (AMP- activated protein kinase) motif at 518-538 aa. Phylogenetically, LrIRAK1 is closely related to cave fish, common carp exhibiting high similarity (~95%) and identity (~90%). In the uninfected fish, the LrIRAK1 expression was highest in liver (~11.5 fold) and lowest in blood. In response to Aeromonas hydrophila, Edwardsiella tarda and Bacillus subtilis infection and various TLR and NLR-ligands stimulation, the expression of LrIRAK1 was markedly enhanced at various time points in almost all the tested tissues. These results together suggest the key role of LrIRAK1 in pattern recognition receptors (PRRs)-mediated host defense against pathogenic insults., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

MyD88 is a universal adapter protein for the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) signaling pathway. Since invertebrates are believed to lack MyD88-independent pathways, MyD88 appears more critical in oyster TLR signaling pathway. In the Pacific oyster (Crassostrea gigas), two complete paralogues, named as CgMyD88-1 and CgMyD88-2, have been identified. In the current study, we indicated that CgMyD88-1 and CgMyD88-2 might act synergistically to increase the efficiency of immune signaling by activating NF-κB transcription factor. However, we found that upon stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid [poly (I:C)], CgMyD88-1 and CgMyD88-2 show differences in their response: CgMyD88-1 accumulated as large spots in the cytoplasm, while CgMyD88-2 assembled in the cytoplasm and in the membrane. Our results support the theory that expansion of these immune genes is associated with functional diversity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this study, we identified a fish-specific Toll-like receptor (TLR) in Pelteobagrus fulvidraco, an economically important freshwater fish in China. This TLR, PfTLR26, was shown to be encoded by a 3084 bp open reading frame (ORF), producing a polypeptide 1027 amino acids in length. The PfTLR26 protein contains a signal peptide, eight leucine-rich repeat (LRR) domains, two LRR&#95;TYP domains in the extracellular region, and a Toll/interleukin (IL)-1 receptor (TIR) domain in the cytoplasmic region, consistent with the characteristic TLR domain architecture. This predicted 117.1 kDa protein was highly homologous to those of other fish, with phylogenetic analysis revealing the closest relation to TLR26 of Ictalurus punctatus. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that the PfTLR26 gene was expressed in all tissues tested, with the highest expression levels seen in the head kidney and blood, and the lowest seen in muscle. PfTLR26 exhibited significant upregulation in liver, spleen, head kidney, and blood at different time points following challenge with the common TLR agonists lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (Poly I:C). Taken together, these results suggest that PfTLR26 may be an important component of the P. fulvidraco innate immune system, participating in the transduction of TLR signaling under pathogen stimulation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) are a category of pattern recognition receptors (PRRs), which recognize pathogen associated molecular patterns (PAMPs) and participate in the immune responses. We identified tlr5a, tlr5b, tlr9 and tlr21 from the genome of blunt snout bream (Megalobrama amblycephala). All four tlrs were constitutively expressed in all examined tissues. After an immune bacterial challenge with Aeromonas hydrophila, their expressionwas up-regulated in lymphoid organs and tissues. Recombinant eukaryotic plasmid pEGFP-N1 was transfected into the common carp (Cyprinus carpio) EPC (epithelioma papulosum cyprini) cells for the purpose of subcellular localization. pcDNA3.1(+) recombinant eukaryotic plasmid was used to investigate the effects of overexpression of tlrs on the expression of downstream interferon-associated immune factors. The four Tlrs were distributed in the cytoplasm of transfected cells and appeared as filamentous or reticular. The expression of irf3, irf7, isg15, mx1, pkr and viperin at 0, 6, 12, 18, 24, 36, 48 and 72 h post-transfection in transfected EPC cells was quantified by qPCR. Overexpression of tlrs upregulated the expression of viperin, isg15, irf3, irf7, mx1 and pkr (in that order of magnitude). We also cloned the following promoters of irfs: Irf1-p, irf2-p, irf6-p, irf7-p, irf8-p and irf9-p. Results of the dual luciferase reporter assay suggested that tlr5a, tlr5b and tlr9 enhanced the activities of irf7-p, while tlr5b enhanced the activities of irf1-p and irf7-p. This suggests that they all play a role in the innate immunity. The experiments also indicated that TLRs activate irf3 or irf7 signaling to induce IFN secretion and subsequent upregulation of IFN-stimulated genes. These results indicate that tlrs and irfs play an important immune role in response to A. hydrophila infection in blunt snout bream, and pave the way for further studies of immune mechanisms mediated by TLRs in fish., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Toll-like receptor (TLR) genes are the earliest reported pathogen recognition receptors (PRRs) and have been extensively studied. These genes play pivotal roles in the innate immune defense against pathogen invasion. In this study, a total of 16 tlr genes were identified and characterized in spotted sea bass (Lateolabrax maculatus). The tlr genes of spotted sea bass were classified into five subfamilies (tlr1-subfamily, tlr3-subfamily, tlr5-subfamily, tlr7-subfamily, and tlr11-subfamily) according to the phylogenetic analysis, and their annotations were confirmed by a syntenic analysis. The protein domain analysis indicated that most tlr genes had the following three major TLR protein domains: a leucine-rich repeat (LRR) domain, a transmembrane region (TM) and a Toll/interleukin-1 receptor (TIR) domain. The tlr genes in spotted sea bass were distributed in 11 of 24 chromosomes. The mRNA expression levels of 16 tlr genes in response to Vibrio harveyi infection were quantified in the head kidney. Most genes were downregulated following V. harveyi infection, while only 5 tlr genes, including tlr1-1, tlr1-2, tlr2-2, tlr5, and tlr7, were significantly upregulated. Collectively, these results help elucidate the crucial roles of tlr genes in the immune response of spotted sea bass and may supply valuable genomic resources for future studies investigating fish disease management., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) play an important role in the activation of innate immune response in animals. In this study, we identified a TLR from the oriental river prawn, Macrobrachium nipponense (MnToll1) and investigated its functions in immunity. The MnToll1 protein shares similar structural characteristics with other known Toll family proteins. MnToll1 transcripts are broadly distributed in all of the examined tissues, and its expression level was significantly up-regulated by bacterial challenge. RNAi-mediated knockdown of MnToll1 significantly impaired the survivability of Vibrio-challenged prawns. RNAi experiments also revealed that the expression of several antimicrobial peptide genes were regulated by MnToll1. Moreover, we found the extracellular region of MnToll1 could directly bind to bacteria and bacterial glycoconjugates. These findings suggest that MnToll1 function as a pattern recognition receptor to recognize invading pathogen and initiate downstream gene expression, to participate in antibacterial defense of M. nipponense., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The white spot syndrome virus (WSSV), the most lethal pathogen of shrimp, is a dsDNA virus with approximately a 300,000 base pairs and contains approximately 180-500 predicted open reading frames (ORFs), of which only 6% show homology to any known protein from other viruses or organisms. Although most of its ORFs encode enzymes for nucleotide metabolism, DNA replication, and protein modification, the WSSV uses some of its encoded proteins successfully to take control of the metabolism of the host and avoid immune responses. The contribution of the shrimp innate immune response to prevent viral invasions is recognized but yet not fully understood. Thus, the role of several components of Toll pathway of the shrimp Penaeus vannamei against WSSV has been previously described, and the consequential effects occurring through the cascade remain unknown. In the current study the effects of WSSV over various components of the shrimp Toll pathway were studied. The gene expression of Spätzle, Toll, Tube, Cactus and Dorsal was altered after 6-12 h post inoculation. The expression of LvToll3, LvCactus, LvDorsal, decreased ~4.4-, ~3.7- and ~7.3-fold at 48, 24 and 48 hpi, respectively. Furthermore, a remarkable reduction (~18-fold) in the expression of the gene encoding LvCactus in WSSV infected specimens was observed at 6 hpi. This may be a sophisticated strategy exploited by WSSV to evade the Toll-mediated immune action, and to promote its replication, thereby contributing to viral fitness., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) play an important role in defense response to pathogens in mollusk. In this study the first TLR from Sepiella japonica (named as SjTLR) was functionally characterized, and its full-length cDNA consisted of 3914bp (GenBank accession no. AQY56780.1) including an open reading frame of 3582bp, encoding a putative protein of 1193 amino acids. Its theoretical molecular weight was 137.87 KDa and the predicted isoelectric point was 3.69. The derived amino acids sequence comprised of an extracellular domain including 26 amino acids signal peptide and eleven leucine-rich repeats (LRR), capped with LRRCT and LRRNT followed by transmembrane domain and cytoplasmic Toll/IL-1R domain (TIR). In addition, 12 potential N-linked glycosylation sites were present in the ectodomain to influence protein trafficking, surface presentation and ligand recognition. Multiple sequence alignment and phylogenetic analysis revealed that SjTLR shared the highest similarity to that of Euprymna scolopes and they fell into the same clade. Real-time PCR showed SjTLR expressed constitutively in all tested tissues, including gill, liver, brain, muscle, intestine, heart, lobus opticus and stomach, but showed different expression levels with genders. The highest expression was in the liver, and the lowest was in stomach for both genders. The functional domain region sequences encoding LRRs domain protein and TIR domain containing protein (TcpB) were expressed in BL21(DE3) respectively and purified with Ni-NAT Superflow resin conforming to the expected molecular weight. The cellular localization of SjTLR in HEK293 cells was conducted and plasma membrane localization was detected. SjLRRs internalization upon the activation of LPS was also observed, and dramatic redistribution of SjLRRs in the cytoplasm with distinct perinuclear accumulation was found. After SjTLR transfection Toll/NF-κB signaling pathway was active in HEK293 treated with LPS and TNFɑ. The nuclear related genes may also be activated by NF-κB in the nucleus, and the corresponding mRNA was transferred through the intracellular signal transduction pathway, so that IL-6 cytokines could be synthesized and released. After infection by Vibrio parahemolyticus and Aeromonas hydrophila the expression of SjTLR were upregulated with time-dependent manner. These findings might be valuable for understanding the innate immune signaling pathways of S.japonica and enabling future studies on host-pathogen interactions., (Copyright © 2019. Published by Elsevier Ltd.)

Toll-like receptors (TLRs) are a large family of pattern recognition receptors (PRRs) that play a critical role in innate immunity. TLRs are activated when they recognize microbial associated molecular patterns (MAMPs) of bacteria, viruses, or fungus. In the present study, two TLRs were isolated from the mantle of the hard-shelled mussel (Mytilus coruscus) and designated McTLR2 and McTLR3 based on their sequence similarity and phylogenetic clustering with Crassostrea gigas, CgiTLR2 and CgiTLR3, respectively. Quantitative RT-PCR analysis demonstrated that McTLR2 and McTLR3 were constitutively expressed in many tissues but at low abundance., (Copyright © 2019. Published by Elsevier Ltd.)

Toll-like receptors (TLRs) are a category of most well recognized pattern recognition molecules that act on a vital role in both innate and adaptive immunity. In the present study, a novel toll-like receptor (McTLRw) was identified and characterized in thick shell mussel Mytilus coruscus. McTLRw possesses one intracellular Toll/interleukin-1 (IL-1) receptor (TIR) domain, one transmembrane region (TM), one leucine rich repeat N-terminal domain (LRR&#95;NT) and a few of leucine-rich repeats (LRRs), which all are common in typical TLRs. McTLRw transcripts were constitutively expressed in all examined tissues with higher expression levels in immune related tissues, and were significantly induced in haemocytes with the challenges of live Vibrio alginolyticus, lipopolysaccharide (LPS), peptidoglycans (PGN) and β-glucan (GLU), but not induced by polyinosinic-polycytidylic acid (poly I:C). rMcTLRw exhibited affinity to LPS, PGN and GLU while no affinity to poly I:C. Further, the downstream of TLR signaling pathway myeloid differentiation factor 88a (MyD88a), interleukin-1 receptor-associated kinase-4 (IRAK4) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were significantly repressed in McTLRw silenced mussels while challenged with LPS. These results collectively indicated that McTLRw is one member of TLR family and involved in immune response to against invaders by taking participate in TLR mediated signaling pathway., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) recognizing specific pathogen-associated molecular patterns play crucial roles in immune defence against pathogen invasion. Although recent advances in many species have reported the characterization and functional roles of TLRs in innate immunity, systematic knowledge of TLRs is still lacking in the Chinese soft-shelled turtle Pelodiscus sinensis. In this study, a genome-wide search was performed and identified 15 candidate PsTLR family genes in P. sinensis. Protein structure analysis revealed the conserved domain arrangements for these PsTLR proteins. Phylogenetic analysis indicated the evolutionary conservation of TLRs among various species. Additionally, a putative interaction network among PsTLR proteins was proposed and several functional partner proteins involved in TLR signalling pathway were predicted in P. sinensis. Expression profiling showed that these PsTLRs exhibited constitutive expression patterns in different tissues of P. sinensis. Moreover, several genes were highly expressed in the major immune organ spleen. Remarkably, the mRNA levels of PsTLR2-1, PsTLR4 and several TLR signalling molecules were significantly up-regulated in the spleen after Aeromonas hydrophila infection, indicating that PsTLRs and these genes responded to bacterial stress. These results provide rich information for the functional exploration of PsTLRs and will facilitate uncovering the molecular mechanisms underlying immune regulation in P. sinensis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Unlike the normal anadromous lifestyle, Chinese native Dolly Varden char (Salvelinus malma) is locked in land and lives in fresh water lifetime. To explore the effect of freshwater adaption on its immune system, we constructed a pooled cDNA library of hepatopancreas and spleen of Chinese freshwater Dolly Varden char (S. malma). A total of 27,829 unigenes were generated from 31,233 high-quality transcripts and 17,670 complete open reading frames (ORF) were identified. Totally 25,809 unigenes were successfully annotated and it classified more native than adaptive immunity-associated genes, and more genes involved in toll-like receptor signal pathway than those in complement and coagulation cascades (51 vs 3), implying the relative more important role of toll-like receptors than the complement system under bacterial injection for the freshwater Dolly Varden char. These huge different numbers of TLR and complement system identified in freshwater Dolly Varden char probably caused by distinct evolution pressure patterns between fish TLR and complement system, representative by TLR3 and TLR5 as well as C4 and C6, respectively, which were under purifying and positively selecting pressure, respectively. Further seawater adaptation experiment and the comparison study with our library will no doubt be helpful to elucidate the effect of freshwater adaption of Chinese native Dolly Varden char on its immune system., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) are well-known pattern recognition receptors that play key immunological roles in a diverse range of organisms. In this study, two novel invertebrate TLRs from disk abalone (designated as AbTLR-A and AbTLR-B) were identified and functionally characterized for the first time. AbTLR-A and AbTLR-B comprised the typical TLR domain architecture containing an extracellular leucine-rich repeat domain, transmembrane domain, and Toll/interleukin-1 receptor domain. Expressional analysis revealed that both TLRs were constitutively expressed at all the early embryonic stages of disk abalone analyzed, with the highest level of AbTLR-A found at the 16-cell stage and AbTLR-B at the trochophore stage. According to tissue distribution analysis, prominent mRNA expression of AbTLR-A and AbTLR-B was detected in the hemocytes and gills, respectively. AbTLR-A and AbTLR-B mRNAs were significantly up-regulated in response to Gram-negative Vibrio parahemolyticus, Gram-positive Listeria monocytogenes, and viral hemorrhagic septicemia virus injections in abalone hemocytes and gills. Overexpression of AbTLR-A and AbTLR-B in HEK293T cells directly activated nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) responsive reporters. Neither TLRs showed a high response to pathogen-associated molecular patterns in vitro. Co-expression of AbTLR-A and AbTLR-B with AbMyD88-2 and AbMyD88-X activated NF-κB-responsive reporters in a synergetic manner. These findings demonstrate the involvement of AbTLR-A and AbTLR-B in abalone innate immunity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

TLR25 is a new member of TLR1 family that is only identified in teleosts, but its function in immune response is still unclear. In current study, the coding sequence (CDS) of TLR25 was cloned from Schizothorax prenanti (named spTLR25), and spTLR25 is 2454 bp in length and coding a protein of 817 aa. The spTLR25 contains a signal peptide, twenty leucine-rich repeat (LRR) domains, a LRR C-terminal (LRRCT) motif, a transmembrane region and a Toll/IL-1 receptor (TIR) domain. Phylogenetic analysis indicates that spTLR25 has the closest relationship with Cyprinus carpio (C. carpio) TLR25-2. The 3D structure of spTLR25 exhibits 5 α-helices and 3 β-sheets in the TIR domain, and 8 α-helices and 6 β-sheets in the LRR domains. The spTLR25 is mainly expressed in immune-related tissues and peripheral blood leukocytes (PBL). Furthermore, the expression levels of spTLR25 were upregulated in spleen, head kidney and liver while S. prenanti was challenged with LPS or Aeromonas hydrophila (Ah), and the upregulation was also detected in head kidney leukocytes (HKL) after LPS and Poly (I:C) stimulation. The luciferase reporter assay demonstrated that NF-κB and type I IFNs signaling pathways can be activated by spTLR25, and this process may involve in the cascade amplification of TLR25-MyD88 signaling. In addition, the co-localization analysis showed that spTLR25 localizes to intracellular region. Taken together, our results reveal that teleost-specific TLR25 may be a multifunctional receptor for recognizing both LPS and Poly (I:C) and may activate NF-κB and type I IFNs signaling pathways., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Dabry's sturgeon (Acipenser dabryanus) is a useful model for the study of fish evolution, as it is one of the most primitive actinopterygian species. However, studies of the immune system of this fish are limited. Here, we identified three toll-like receptors (adaTLR21, adaTLR22, and adaTLR25) from Dabry's sturgeon. The three sturgeon TLRs had characteristic TLR features, including a signal peptide, several leucine rich repeat (LRR) domains, a transmembrane domain, and a Toll/interleukin-1 receptor (TIR) domain. Although the predicted amino acid sequences encoded by the sturgeon adaTLR21, adaTLR22, and adaTLR25 had somewhat low levels of sequence identity and similarity with TLRs from other fish species, the three sturgeon TLRs fell in well-supported clades with other teleost TLRs in our neighbor-joining phylogenetic tree. Real-time quantitative PCR showed that the three sturgeon TLRs were ubiquitously expressed in all examined tissues from healthy adult sturgeon, but that their expression patterns varied greatly among the different tissues. The three sturgeon TLRs were also expressed across all embryonic developmental stages that were examined, but their expression levels differed between developmental stages. All three TLRs were upregulated in head-kidney primary leucocytes following lipopolysaccharide (LPS) and polyinosinic: polycytidylic acid (polyI:C) stimulation. To the best of our knowledge, this is the first characterization of these three TLRs in Darby's sturgeon. Our results provide a framework for further studies of TLR ligand specificity and signaling pathways in sturgeon, and increase our understanding of the functional evolution of TLRs in vertebrates., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adapter molecules in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. Despite of the well study in vertebrates, there is few data ascribe to this TRAF member in invertebrates, especially in bivalves. In the present study, a novel TRAF6 homologue termed McTRAF6 was firstly characterized in Mytilus coruscus. Like its counterparts in mammals, McTRAF6 shared the domain topology containing one RING domain, two zinc finger domains, one coiled-coil region and a MATH domain. McTRAF6 transcripts predominantly expressed in gills, digestive glands and hemocytes in M. coruscus, and were significantly up-regulated in hemocytes after challenge with lipopolysaccharide (LPS) and polyinosine-polycytidylic acid (poly I:C). Further, the subcellular localization in cytoplasm and the activation of Nk-κB or ISRE luciferase reporter by overexpressed McTRAF6 were identified in HEK293T cells. These results collectively indicate that McTRAF6 is a member of TRAF6 subfamily and plays a potential role in immune defense system against pathogenic agents invasions in thick shell mussel. To our knowledge, this is the first report on component of TLR signaling pathway in thick shell mussel, providing further evidence for the existence of TLR pathway in M. coruscus and contribute to clarify the innate immune system of thick shell mussel., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Toll-like receptor (TLR) is considered to be an evolutionarily conserved transmembrane protein which promotes the Toll signal pathway to active the expression of transcription factors in the innate immunity of the organism. In this study, a full length of TLR homologue of 2525bp in Mytilus coruscus (named as McTLR-a, GenBank accession no: KY940571) was characterized. Its ORF was 1815 bp with a 5'untranslated region (UTR) of 128 bp and a 3'UTR of 582 bp, encoding 602 amino acid residues with a calculated molecular weight of 70.870 kDa (pI = 6.10). BLASTn analysis and phylogenetic relationship strongly suggested that this cDNA sequence was a member of TLR family. Quantitative real time RT-PCR showed that constitutive expression of McTLR-a was occurred, with increasing order in hemocyte, gonad, mantle, adducter, gill and hepatopancreas. Bacterial infection and heavy metals stimulation up-regulated the expression of McTLR-a mRNA in hepatopancreas with time-dependent manners. The maximum expression appeared at 12 h after pathogenic bacteria injection, with approximately 22-fold in Aeromonas hydrophila and 17-fold in Vibrio parahemolyticus higher than that of the blank group. In heavy metals stress group, they all reached peaks at 3d, while the diverse concentration caused the maximum expression were different. The highest expression reached approximately 7-fold higher than the blank in low concentration of Pb 2+ exposure. In Cu 2+ treated group, it reached the peak (approximately 12-fold higher than the blank)in middle concentration. These results indicated that McTLR-a might be involved in the defense response and had a significant role in mediating the environmental stress., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The innate immune system is the first line defense mechanism that recognizes, responds to, controls or eliminates invading pathogens. Toll-like receptors (TLRs) are a critical family of pattern recognition receptors (PRRs) tightly regulated by complex mechanisms involving many molecules to ensure a beneficial outcome in response to foreign invaders. MicroRNAs (miRNAs), a transcriptional and posttranscriptional regulator family in a wide range of biological processes, have been identified as new molecules related to the regulation of TLR-signaling pathways in immune responses. To date, at least 22 TLR types have been identified in more than a dozen different fish species. However, the functions and underlying mechanisms of miRNAs in the regulation of inflammatory responses related to the TLR-signaling pathway in fish is lacking. In this review, we summarize the regulation of miRNA expression profiles in the presence of TLR ligands or pathogen infections in teleost fish. We focus on the effects of miRNAs in regulating TLR-signaling pathways by targeting multiple molecules, including TLRs themselves, TLR-associated signaling proteins, and TLR-induced cytokines. An understanding of the relationship between the TLR-signaling pathways and miRNAs may provide new insights for drug intervention to manipulate immune responses in fish., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) are one of the most important innate immune receptors, which recognize various pathogen-associated molecular patterns and activate the downstream immune response. Mouse TLR13 has been found to recognize a highly conserved sequence from bacterial or viral RNA and activate the myeloid differentiation primary response gene 88-dependent signaling response. The function of teleost tlr13 is still not fully understood, especially its relationship with bacterial RNA. In our study, we identified and characterized a tlr13 from Epinephelus coioides (orange-spotted grouper). The full-length cDNA of Eco. tlr13 contained a 2844 bp open reading frame, encoding 947 amino acids. The polypeptide was constitutive of a signal peptide, 13 leucine-rich repeats domains, a C-terminal leucine-rich repeats, a transmembrane domain and a conserved Toll/interleukin (IL)-1 receptor domain, indicating that Eco. Tlr13 exhibited a typical TLR structure. Multiple alignments showed that the Toll/IL-1 receptor domain of Eco. Tlr13 was identical with other homologues, and the phylogenetic tree suggested that Eco. Tlr13 was clustered with other TLR13s and had the closest relationship with predicted Lates calcarifer (sea bass) Tlr13. Subcellular localization analysis revealed that Eco. Tlr13 colocalized with the endoplasmic reticulum and early endosome. Moreover, Eco. tlr13 was broadly observed in all tested tissues with the relatively high expressions in the brain and immune-related tissues. After challenged with 19-mer Staphylococcus aureus 23S ribosomal RNA-derived oligoribonucleotide (ORN Sa19), the expression of Eco. tlr13 was significantly up-regulated in grouper spleen cells. Also, the luciferase assay further revealed that with the overexpression of Eco. Tlr13 in human embryonic kidney 293T cells, ORN Sa19 activated the promoter activity of interferon-β in a dose-dependent pattern. These results indicate that Eco. tlr13 may involve in the recognition of bacterial RNA., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Toll-like receptors are important pattern recognition receptors that can recognize pathogen-associated molecular patterns (PAMPs) and play a critical role in innate immunity. In the present study, tlr18 was identified from common carp (Cyprinus carpio L.) (named Cctlr18). The deduced amino acid sequence contained only a signal peptide, eight LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/IL-1 receptor) domain. Phylogenetic analysis showed that CcTlr18 was most closely related to Ctenopharyngodon idella Tlr18. Quantitative real-time PCR analysis showed that Cctlr18 was constitutively expressed in all investigated tissues with the highest expression level in the skin and lowest expression in the gonad. After injection with inactivated Aeromonas hydrophila, Cctlr18 expression was significantly up-regulated in the head kidney, foregut, hindgut and skin. Moreover, significant up-regulation of Cctlr8 was observed in the spleen, head kidney, hindgut and skin after immersion with live A. hydrophila. In addition, the expression of Cctlr18 was up-regulated in PGN or flagellin-stimulated HKLs. Luciferase reporter assays showed that Cctlr18 activated NF-κB in 293 T cells and that NF-κB activity was enhanced in Cctlr18 and Ccmyd88 co-transfected cells. Furthermore, Cctlr18 could induce the expression of cytokines genes, including ifn, il-1β and il-10, in EPC cells. The results suggested that Cctlr18 plays an important role in the immune response and provides basic information for investigating the mechanisms of fish tlr18., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

TLR22, a typical member of the fish-specific TLRs, is a crucial sensor in virally triggered innate immune signalling retained from natural selection. To elucidate the role of the TLR22-specific signalling cascade mechanism, we provide evidence that the double-stranded (ds) RNA-sensor TLR22 positively regulates the ERK pathway and negatively regulates the JNK, p38 MAP kinase and NF-κB pathway. Here, we show that TLR22 restrains NF-κB activation and IFN (interferon) β and AP-1 (activator protein-1) promoter binding (impairing "primary response" genes (TNF and IL-1)), induces "secondary response" genes (IL-12 and IL-6) and mediates the irregular expression of inflammatory genes. Therefore, TLR22 promotes ERK phosphorylation but impairs the JNK and p38 MAP kinases and IκB phosphorylation. Additionally, TLR22 controls the excessive generation of reactive oxygen species (ROS) to avoid damaging the organism. The specific kinetics of TLR22 depends on its distinct cellular localization. We demonstrate that TLR22 is an intracellular receptor localized in the endosome, and the TLR22-TIR domain is the functional structure inducing the signalling cascade post-viral replication in the body. As mentioned above, our data reveal a novel mechanism whereby TLR22-induced positive adjustment and negative regulation evolved independently to avoid harmful and inappropriate inflammatory responses., (Copyright © 2017. Published by Elsevier Ltd.)

Toll-like receptors, the best known pattern recognition receptors, play important roles in recognizing non-self molecules and binding pathogen-associated molecular patterns in the innate immune system. In the present research, the cDNA and protein characterization of the TLR signalling pathway genes including IRAK4, TRAK6 and IKKα (named CsIRAK4, CsTRAF6 and CsIKKα, respectively) with the typical motifs from Cyclina sinensis showed significant similarity with their homologues from other shellfish. Furthermore, the mRNA transcripts of these three genes are ubiquitously expressed in all tissues tested and are dominantly expressed in C. sinensis haemocytes (P < 0.05). Moreover, IRAK4, TRAK6 and IKKα cDNA expression levels were all up-regulated after injection with Vibrio anguillarum, Micrococcus luteus and poly I:C (P < 0.01) as shown by quantitative real-time PCR, indicating that they were involved in responding to pathogenic stimulation. We explored the function of the TLR13-MyD88-NF-κB signalling pathway in the innate immune responses of C. sinensis by RNA interference and immune challenges. The results suggested the mRNA expression patterns of CsMyD88, CsIRAK4, CsTRAF6, CsIKKα, CsIκB, CsNF-κB, CsC-LYZ and CsAMP were all down-regulated (P < 0.01) in normal and stimulated C. sinensis haemocytes, revealing the involvement of the TLR13-MyD88-NF-κB signalling pathway in innate immunity by positively adjusting internal signalling factors and immune-related genes. In summary, a TLR13-MyD88-NF-κB signalling pathway exists and plays vital roles in innate immune responses in C. sinensis. These findings collectively lay the foundation for studying the functional characterization of internal signalling factors and establishing a regulatory network for the TLR signalling pathway in molluscs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) play a crucial role in innate immunity by recognizing specific pathogen-associated molecular patterns, including lipoproteins, lipopeptides, lipopolysaccharide, flagellin, dsRNA, ssRNA and CpG DNA motifs. Although significant effects of TLRs on immunity have been reported in most vertebrates and some invertebrates, the complete TLR superfamily has not been systematically characterized in scallops. In this study, 18 TLR genes were identified from Yesso scallop (Patinopecten yessoensis) using whole-genome scanning. Phylogenetic and protein structural analyses were performed to determine the identities and evolutionary relationships of the 18 genes. Extensive expansion of TLR genes from the Yesso scallop genome indicated gene duplication events. In addition, expression profiling of PyTLRs was performed at different acidifying exposure levels (pH = 6.50, 7.50) with different challenge durations (3, 6, 12 and 24 h) via in silico analysis using transcriptome and genome databases. Our results confirmed the inducible expression patterns of PyTLRs under acidifying exposure, and the responses to immune stress may have arisen through adaptive recruitment of tandem duplications of TLR genes. Collectively, this study provides novel insight into PyTLRs as well as the specific role and response of TLR signaling pathways in host immune responses against acidifying exposure in bivalves., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) are one of the most extensively researched pattern recognition receptors (PRRs) and play an important role in the innate immune system. In this study, partial cDNA sequences of the Pf&#95;TLR18 and Pf&#95;TLR19 genes and complete cDNA sequence of the Pf&#95;TLR21 gene were cloned from yellow catfish (Pelteobagrus fulvidraco). The open reading frames (ORFs) of the Pf&#95;TLR18, Pf&#95;TLR19 and Pf&#95;TLR21 genes were 1956 bp, 2262 bp and 2949 bp in length, encoding 651, 753 and 982 amino acids, respectively. The Pf&#95;TLR18 and Pf&#95;TLR19 consist of leucine-rich repeats (LRRs), a transmembrane domain and a Toll/interleukin-I receptor domain, and the Pf&#95;TLR21 only has LRRs and TIR domain. Homologous identity revealed that the Pf&#95;TLR18, Pf&#95;TLR19 and Pf&#95;TLR21 genes have high nucleotide and protein sequence similarity with channel catfish, especially the TIR domains that exhibited the greatest conservation compared to channel catfish. Ontogenetic expression analyses indicated that the mRNA expressions of the Pf&#95;TLR18, Pf&#95;TLR19 and Pf&#95;TLR21 genes could be detected from fertilized eggs to 30 day post-hatching and they exhibited different variation trends after hatching. The three TLR genes were expressed in various tissues, but they were mostly highly expressed in the spleen. The mRNA expression levels of the three genes were up-regulated in the spleen, head kidney, trunk kidney, liver and blood after challenge of killed Aeromonas hydrophila. In addition, the expressions of the three TLR genes were induced to up-regulate in isolated peripheral blood lymphocytes of yellow catfish after stimulation with lipopolysaccharides (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (Poly I:C). Our findings indicate that the three TLR genes may play a potential role in the host defense against pathogenic microbes. These results will provide valuable information to better understand the function of TLR genes in the innate immune system of yellow catfish., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Toll-like receptor 21 (TLR21) is a non-mammalian TLR that functions similar to mammalian TLR9 in recognizing CpG DNA. In the present study, we identified a TLR21 homologue, LycTLR21, from large yellow croaker (Larimichthys crocea). The complete coding sequence of LycTLR21 is 2946 nucleotides long, encoding a protein of 981 amino acids. The deduced LycTLR21 protein has typical TLR domain architecture, including a signal peptide, 13 leucine-rich repeats (LRRs) in the extracellular region, a transmembrane region, and a cytoplasmic Toll-Interleukin-1 receptor (TIR) domain. Phylogenetic analysis showed that LycTLR21 falls into a major clade formed by all fish TLR21 sequences and is closely related to TLR21 in Epinephelus coioides and Oplegnathus fasciatus. LycTLR21 mRNA was constitutively expressed in all tissues tested, with higher levels in immune-related tissues, such as spleen, head kidney, and gills. Upon stimulation with inactivated trivalent bacterial vaccine, LycTLR21 mRNA was significantly increased in these three tissues. Overexpression of a chimeric plasmid containing the extracellular domain of human cluster of differentiation 4 (CD4) and the transmembrane and cytoplasmic domains of LycTLR21 could activate NF-κB, but not IFN-β in Chinese hamster ovary (CHO) cells, suggesting that LycTLR21 could mediate activation of NF-κB. LycTLR21 could specifically recognize three CpG-oligodeoxynucleotides (CpG-ODNs), CpG-ODN 1826, 2006, and 2007, but not other CpG-ODNs detected, poly(I:C), lipopolysaccharide (LPS), and lipoteichoic acid (LTA-SA). These three CpG-ODNs were found to significantly up-regulate the expression of LycTLR21 and downstream proinflammatory cytokines IL-1β and IL-6 of NF-κB pathway in large yellow croaker head kidney (LYCK) cells. In addition, the expression levels of LycTLR21, c-Rel subunit of NF-κB, IL-1β and IL-6 genes were quickly increased in the spleen and head kidney by bacterial infection, suggesting that LycTLR21 signaling pathway may play a role in immune response to bacterial infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLR) are key components of innate immunity that play significant roles in immune defense against pathogens invasion. Recent frequent outbreaks of the "white spot disease" caused by parasitic infection in farmed Tibetan fishes had resulted in great economic losses. However, to our knowledge, the roles of TLRs in mediating immune response to parasitic infection in Tibetan fishes remain to be determined. Here, we performed data-mining on a widely-farmed Tibetan fish (Gymnocypris przewalskii or Gp) transcriptome to determine the genetic variation and expression pattern of TLRs. We totally obtained 14 GpTLRs and identified 5 with a complete coding sequence. Phylogenetic analysis verified their identities and supported the classification of TLRs into six families as in other vertebrates. The TLR family motifs, such as leucine rich repeat (LRR) and Toll/interleukin (IL)-1 receptor (TIR) domain, are conserved in GpTLR1-5. Selective pressure test demonstrated that all known GpTLRs are under purifying selection, except GpTLR4 underwent positive selection. Further, site model analysis suggested that 11 positively selected sites are found in LRR domain of GpTLR4. Three positively selected sites are located on outside surface of TLR4 3D structure, indicating that function of GpTLR4 may be affected. Tissue specific expression analysis showed all GpTLRs are present in gill, head-kidney and spleen but the relative abundance varied among tissues. In response to parasite Ichthyophthirius multifiliis infection, 5 GpTLR (GpTLR1, -2, -4, -9 and -20) expressions were induced. Intriguingly, GpTLR4 was significantly up-regulated in gills, while GpTLR19 and GpTLR21 unexpectedly showed no any change. In summary, these results revealed the first genomic resources of TLR family and several parasitic infection responsive TLRs in Tibetan fish. These findings provide key information for future studies aiming to understand the molecular mechanisms underlying the immune response to pathogen invasion in Tibetan fishes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Pattern recognition receptors (PRRs) recognize the conserved molecular structure of pathogens and trigger the signaling pathways that activate immune cells in response to pathogen infection. Toll-like receptors (TLRs) are the first and best characterized innate immune receptors. To date, at least 20 TLR types (TLR1, 2, 3, 4, 5M, 5S, 7, 8, 9, 13, 14, 18, 19, 20, 21, 22, 23, 24, 25, and 26) have been found in more than a dozen of fish species. However, of the TLRs identified in fish, direct evidence of ligand specificity has only been shown for TLR2, TLR3, TLR5M, TLR5S, TLR9, TLR21, and TLR22. Some studies have suggested that TLR2, TLR5M, TLR5S, TLR9, and TLR21 could specifically recognize PAMPs from bacteria. In addition, other TLRs including TLR1, TLR4, TLR14, TLR18, and TLR25 may also be sensors of bacteria. TLR signaling pathways in fish exhibit some particular features different from that in mammals. In this review, the ligand specificity and signal pathways of TLRs that recognize bacteria in fish are summarized. References for further studies on the specificity for recognizing bacteria using TLRs and the following reactions triggered are discussed. In-depth studies should be continuously performed to identify the ligand specificity of all TLRs in fish, particularly non-mammalian TLRs, and their signaling pathways. The discovery of TLRs and their functions will contribute to the understanding of disease resistance mechanisms in fish and provide new insights for drug intervention to manipulate immune responses., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Disease is caused by a complex interaction between the pathogen, environment, and the physiological status of the host. Determining how host ontogeny interacts with water temperature to influence the antiviral response of the Pacific oysters, Crassostrea gigas, is a major goal in understanding why juvenile Pacific oysters are dying during summer as a result of the global emergence of a new genotype of the Ostreid herpesvirus, termed OsHV-1 μvar. We measured the effect of temperature (12 vs 22 °C) on the antiviral response of adult and juvenile C. gigas injected with poly I:C. Poly I:C up-regulated the expression of numerous immune genes, including TLR, MyD88, IκB-1, Rel, IRF, MDA5, STING, SOC, PKR, Viperin and Mpeg1. At 22 °C, these immune genes showed significant up-regulation in juvenile and adult oysters, but the majority of these genes were up-regulated 12 h post-injection for juveniles compared to 26 h for adults. At 12 °C, the response of these genes was completely inhibited in juveniles and delayed in adults. Temperature and age had no effect on hemolymph antiviral activity against herpes simplex virus (HSV-1). These results suggest that oysters rely on a cellular response to minimise viral replication, involving recognition of virus-associated molecular patterns to induce host cells into an antiviral state, as opposed to producing broad-spectrum antiviral compounds. This cellular response, measured by antiviral gene expression of circulating hemocytes, was influenced by temperature and oyster age. We speculate whether the vigorous antiviral response of juveniles at 22 °C results in an immune-mediated disorder causing mortality., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The interleukin 1 receptor-associated kinase 4 (IRAK4) is an essential factor for TLR-mediated activation of the host's immune functions subsequent to pathogen contact. We have characterized the respective cDNA and gene sequences from three salmonid species, salmon, rainbow trout and maraena whitefish. The gene from salmon is structured into eleven exons, as is the mammalian homologue, while exons have been fused in the genes from the two other salmonid species. Rainbow trout expresses also a pseudogene at low levels. Its basic structure resembles more closely the primordial gene than the functional copy does. The N-terminal death domain and the C-terminal protein kinase domain of the factors are better conserved throughout evolution than the linker domain. The deduced amino acid sequences of the factors from all three species group together in an evolutionary tree of IRAK4 factors. Scrutinizing expression and function of IRAK4 from rainbow trout, we found its highest expression in head kidney and spleen and lowest expression in muscle tissue. Infecting fish with Aeromonas salmonicida did not modulate its expression during 72 h of observation. Expression of a GFP-tagged trout IRAK4 revealed, expectedly, its cytoplasmic localization in human HEK-293 cells. However, this factor significantly quenched in a dose-dependent fashion not only the pathogen-induced stimulation of NF-κB factors in the HEK-293 reconstitution system of TLR2 signaling, but also the basal NF-κB levels in unstimulated control cells. Our data unexpectedly imply that IRAK4 is involved in establishing threshold levels of active NF-κB in resting cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)